CN114085908A

CN114085908A - Gene target combination for evaluating treatment effect of glioblastoma multiforme and application thereof

Info

Publication number: CN114085908A
Application number: CN202111278585.XA
Authority: CN
Inventors: 王岩; 何江; 卞修武
Original assignee: First Affiliated Hospital of Army Medical University
Current assignee: First Affiliated Hospital of Army Medical University
Priority date: 2021-10-30
Filing date: 2021-10-30
Publication date: 2022-02-25
Anticipated expiration: 2041-10-30
Also published as: CN114085908B

Abstract

The invention provides a gene target combination for evaluating the treatment effect of glioblastoma, which comprises a CCL2 gene and a LIF gene. The invention also provides application of the diagnostic reagent and a diagnostic reagent for evaluating the treatment effect of the glioblastoma. The gene target combination and the diagnostic reagent based on the invention can effectively assist clinical detection and evaluation of prognosis and malignant characterization of mGBM.

Description

Gene target combination for evaluating treatment effect of glioblastoma multiforme and application thereof

Technical Field

The invention relates to the technical field of diagnostic reagents, in particular to a gene target combination for evaluating the treatment effect of glioblastoma multiforme and a diagnostic reagent.

Background

Glioblastoma (GBM) is the most common primary malignancy in the adult brain, with 10% -20% of GBM patients diagnosed with more than one tumor lesion or multifocal GBM (mGBM) the overall survival of mGBM patients is shortened compared to patients with only one GBM mass or a single lesion GBM (ugbm), resistant to current therapeutic measures.

GBMs can be classified into 4 molecular subtypes based on transcriptomic characteristics and genetic alterations, namely, Proneural (Neural), Neural (Neural), Classical (classic) and Mesenchymal (mesenchyme) subtypes, with distinct genetic and transcriptomic characteristics, respectively. In clinical prognosis and treatment, 4 subtypes also respond to different survival lengths and treatment responses. The reports show that the Mesenchymal GBM is most malignant and has the strongest tolerance to conventional chemoradiotherapy, and the regulation mechanism is not clear.

CCL2 is designated as C-C motif chemokine ligand 2, and is referred to herein as cysteine-cysteine motif chemokine ligand 2. LIF is called Leukemia inhibition Factor or LIF Interleukin 6Family Cytokine, and is named as Leukemia Inhibitory Factor or LIF Interleukin 6Family Cytokine. Previous studies have reported that they play a role in the immunomodulation process.

Given that the prognosis of mGBM is worse than uGBM, determining key genes involved in GBM progression may reveal underlying mechanisms of poor GBM prognosis and providing new therapeutic targets for mGBM and uGBM is a current problem that is urgently needed to solve.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an application of combining CCL2 and LIF expression conditions to evaluate prognosis and drug resistance characteristics of glioblastoma multiforme patients, which is a problem that workers in the field need to solve urgently, and can provide more diversified and personalized means for clinical diagnosis and treatment.

The invention firstly provides a gene target combination for evaluating the treatment effect of glioblastoma, which comprises a CCL2 gene and a LIF gene.

The invention further provides application of the gene target combination in preparing a diagnostic reagent for evaluating the treatment effect of the glioblastoma.

In one embodiment of the invention, the expression of CCL2 gene and LIF gene is detected.

In one embodiment according to the present invention, the expression of CCL2 gene and LIF gene is determined by measuring mRNA or protein corresponding to the expression of CCL2 gene and LIF gene.

In one embodiment according to the present invention, the clinical prognosis or treatment tolerance of glioblastoma is determined.

Another aspect of the present invention provides a diagnostic reagent for evaluating the effect of glioblastoma treatment, comprising a preparation for determining the expression of CCL2 gene and LIF gene.

In one embodiment according to the invention, the agent is selected from one or more of a primer, a probe or an antibody.

The invention has the following beneficial effects:

the invention provides an application of combining the expression conditions of CCL2 and LIF to the evaluation of prognosis and drug resistance characteristics of a glioblastoma patient, and the evaluation of prognosis and malignant characterization of mGBM can be effectively assisted with clinical detection.

Drawings

FIG. 1 is a graph relating the expression and clinical significance of LIF and CCL2 in GBM; wherein the content of the first and second substances,

a is representative Immunohistochemistry (IHC) image of CCL2 and LIF protein marker expression on three tumor foci of patient specimen mGBM1_ a-C, scale bar: 100 microns;

b is a mRNA expression profile for detecting LIF and CCL2 in GBM of three different molecular subtypes, namely, a Proneural subtype (neural), a Classical subtype (classic) and a Mesenchymal subtype (mesenchyme) by using a TCGA _ GBM database;

c is a percentage map of four molecular subtypes in the GBM sample of LIFHIgh/CCL2High and the GBM sample of LIFLow/CCL2Low, wherein the Mesenchymal subtype (Mesenchymal) in the GBM sample of LIFHIgh/CCL2High accounts for the highest percentage, and the Proneural subtype (Proneural) in the GBM sample of LIFLow/CCL2Low accounts for the highest percentage.

d is a representative IHC image of CCL2 and LIF protein marker expression on glioma tissue microarray, scale bar: 100 microns;

e is a patient survival curve analysis map based on LIF or/and CCL2 expression in a glioma tissue chip sample and a TCGA _ GBM database;

f is a graph of the protein levels of LIF and CCL2 in the sera of 2 mGBM patients (mGBM1 and mGBM2) and another 5 uGBM patients determined by ELISA.

FIG. 2 is a correlation plot of LIF/CCL2 co-expression, mGBM and mesenchymal subtype GBM; wherein the content of the first and second substances,

a is a GSEA enrichment analysis map of Verhaak _ Glioblastma _ Mesenchymal gene set for GBMs with different phenotypes in a TCGA _ GBM database;

b is a map for performing GSEA enrichment analysis on GBMs with different phenotypes in a TCGA _ GBM database by using a Hallmark _ Epithelial _ Mesenchymal _ Transition gene set;

c is transcriptome clustering heatmap of different GBM phenotypes obtained from TCGA _ GBM database by GSEA analysis.

d is an enriched Verhaak _ Glioblastoma _ Mesenchymal gene set map of LN18 cells treated with LIF/CCL2 and vehicle;

e is an enriched Hallmark _ Epithelial _ Mesenchyl _ Transition gene set map of LN18 cells treated with LIF/CCL2 and vehicle;

f is a venn plot of LIF/CCL 2-treated versus vehicle-treated LN18 cells significantly upregulated genes, and mesenchymal subtypes in the TCGA _ GBM database versus other subtypes, FC representing fold change;

g is a schematic representation of tumor initiation and progression for mGBM.

FIG. 3 is a map relating the expression and prognosis of mGBM1 different molecular subtype characteristic genes; wherein the content of the first and second substances,

c is a Pearson correlation analysis profile between the mRNA expression of LIF and the mRNA expression of CCL2 in different public databases of GBM;

d is a graph for detecting the mRNA expression condition of LIF and CCL2 in different molecular subtypes by using 3 GBM databases;

f is a Kaplan-Meier survival curve analysis map based on the LIF or CCL2 expression quantity in three GBM databases;

g is Kaplan-Meier survival analysis map of LIFHigh/CCL2High and LIFLow/CCL2Low or combination of mesenchymal subtype and proneural subtype patients in TCGA _ GBM database (left) or single-scheme (right) treatment.

Fig. 4 is a GSEA analysis of different GBM phenotypes from the TCGA _ GBM database. a) -d) wien plots of different GBM phenotype enriched gene sets in the context of the indicated gene set.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

Unless otherwise stated, the instruments and materials referred to in the following examples are commercially available.

Unless otherwise stated, reagents referred to in the following examples were commercially available analytical grade reagents.

Antibodies and reagents

anti-CCL 2(# sc-32771) was from Santa Cruz Biotechnology.

anti-LIF (# ab138002) is from abcam.

Recombinant human LIF (#300-05) and recombinant human CCL2(#300-04) were from PeproTech.

Cell culture

Human GBM cell line LN18 was from ATCC (www.ATCC.org) and contained 5% CO at 37 ℃₂The culture was performed in the incubator of (1), and the basic medium used was DMEM/F12 supplemented with 10% fetal bovine serum. This cell line was characterized by STR DNA fingerprinting using the AmpFLSTR identifier kit (#4322288, ThermoFisher) half a year according to the manufacturer's instructions and verified as mycoplasma negative. STR profiles are compared to known ATCC fingerprints and can be matched to known DNA fingerprints. LIF and CCL2 recombinant proteins were added according to the manufacturer's product data sheet and associated instructions.

Example 1 transcriptome sequencing analysis the relevance of LIF/CCL2 to Mesenchymal typing and mGBM

1) Library preparation for transcriptome sequencing

Total cellular RNA was extracted using TRIzol reagent (15596026ThermoFisher, USA) according to the manufacturer's instructions. A total of 2. mu.g of RNA per sample preparation was used as RNA sample input material. Use of

Is/are as follows

UltraTM RNA library preparation kit (NEB, USA) generates sequencing libraries and adds an index code to each sampleIn the attribute sequence of (2). In the general procedure, mRNA was purified from total RNA using poly-T oligonucleotide-linked magnetic beads. Samples were fragmented in NEBNext first strand synthesis reaction buffer (5X) by using divalent cations at high temperature. The first strand of the cDNA was synthesized using random hexamer primers and M-MuLV reverse transcriptase (RNase H-). Second strand cDNA synthesis was then performed using DNA polymerase I and RNase H. The remaining single stranded overhang is converted to blunt ends by exonuclease/polymerase activity. After adenylation of the 3' -end of the DNA fragment, a NEBNext linker having a hairpin loop structure was ligated to prepare hybridization. To screen cDNA fragments preferentially 150-200 bp in length, library fragments were purified using the AMPure XP system (Beckman Coulter, Beverly, USA). Then, the linker-ligated cDNA was reacted at 37 ℃ for 15min, and then reacted at 95 ℃ for 5min, followed by PCR. PCR was then performed using Phusion high fidelity DNA polymerase, universal PCR primer set and index (X) primers. Finally, the PCR products were purified (AMPure XP system) and the quality of the library was assessed on the Agilent Bioanalyzer 2100 system.

2) Clustering and sequencing

Index-coding samples were clustered on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the manufacturer's instructions. After cluster generation, the library preparations were sequenced on the Illumina Hiseq platform to generate 125bp/150bp paired-end reads.

3) Quality control

Raw data in fastq format (raw read) is first processed by an internal perl script. In this step, clean data (clean read) is obtained by deleting the read containing the adapter, the read containing ploy-N, and the low quality read from the original data. At the same time, the Q20, Q30, and GC contents of the pure data were calculated. All downstream analyses were based on high quality clean data.

4) Mapping reads to a reference genome

The reference genome and gene model annotation files are downloaded directly from the genome website. The reference genome was indexed using Bowtie v2.2.3 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Hisat2 was chosen as the mapping tool here because Hisat2 can generate a database of splice sets based on gene model annotation files, with better mapping results than other non-splicing mapping tools.

5) Quantification of gene expression levels

HTSeq was used to count the number of reads mapped to each gene. FPKM for each gene was then calculated based on the length of the gene and the number of fragments mapped to the gene was read. FPKM represents the fragment of each million mapping reads per kilobase of transcription, taking into account the effects of sequencing depth and gene length on reads count, is currently the most commonly used method to estimate gene expression levels.

6) Differential expression analysis

Differential expression analysis was performed using the edgeR software package. edgeR is one of the most popular Bioconductor packages to assess differential expression in RNA-seq data. It is based on Negative Binomial (NB) distribution and models the variation between biological replications by NB discrete parameters. This approach immediately enables the processing of complex experimental designs. Genes found by DESeq with a corrected P value <0.05 were defined as differentially expressed. Differential expression analysis of the two conditions was performed using the DEGSeq R software package. The P value was adjusted using the Benjamini & Hochberg method. Corrected P values of 0.005 and log2 (fold change) to 1 were set as thresholds for significant differential expression.

As can be seen in FIG. 2d, treatment of LN18 cells with LIF/CCL2 resulted in cell enrichment with the Verhaak _ Glioblastoma _ Mesenchymal gene set profile, indicating that expression of LIF/CCL2 contributes to the phenotypic characteristics of Mesenchymal in glioma cells. FIG. 2e illustrates that treatment of LN18 cells with LIF/CCL2 enriches the Hallmark _ Epithelial _ Mesenchymal _ Transition gene set profile, and that LIF/CCL2 expression promotes the transformation of cells into the Mesenchymal phenotype. While in a common database, such as the LIF shown in FIG. 2a^High/CCL2^HighAnd both the Mesenchymal phenotype and the mGBM can also enrich the Verhaak _ Glioblastma _ Mesenchymal gene set map. Similarly, FIG. 2b shows LIF in public databases^High/CCL2^HighAnd Mesenchymal phenotype and mGBM can enrich Hallmark _ episeal _ mesenchyme _ Transition gene set map. Further by gene expression clustering analysis as shown in FIG. 2c, we can find LIF^High/CCL2^HighAnd Mesenchymal phenotype and mGBM can be grouped into one class (first three columns), while LIF^Low/CCL2^LowAnd Non-Mesenchymal phenotype and uGBM into another class (the last three columns). LIF was shown by analysis of different gene sets^H/CCL2^H vs.LIF^L/CCL2^LThe differential gene set of (a), the differential gene set of Mesenchymal vs. other, and the differential gene set of mGBM vs. uGBM, there are a large number of intersections between them, as shown in FIGS. 4 a-d.

While LIF/CCL2 was also found to be highly expressed in the Mesenchymal subtype in public databases as shown in FIGS. 1b and 3d, FIG. 1c also shows that in LIF^H/CCL2^HThat is, the percentage of Mesenchymal typing in the samples with high expression of both LIF and CCL2 reached 55.6%, while LIF^L/CCL2^LThat is, the percentage of mesenchyme typing in samples where both LIF and CCL2 were under-expressed was only 7.2%.

The results in fig. 2f show that significant upregulation genes in LIF/CCL 2-treated LN18 cells partially overlapped with mesenchymal subtypes in the TCGA _ GBM database compared to other subtypes, demonstrating that LIF/CCL 2-treatment induced the expression of partially mesenchymal subtype-specific expressed genes.

The above results and conclusions indicate that LIF^High/CCL2^HighThe method has great correlation with the Mesenchymal phenotype and the mGBM, the similarity of gene expression profiles of the Mesenchymal phenotype and the mGBM is extremely high, and a large amount of overlap exists, so a tumor evolution model is provided, as shown in figure 2g, the initial uGBM focus formed by the tumor is gradually induced to form a more malignant Mesenchymal subtype under the action of tumor microenvironment factors such as LIF/CCL2, and the initiation and the progress of the mGBM are promoted.

Example 2 TMZ IC50 calculation, and analysis of prognostic data, verifies that LIF/CCL2 promotes treatment tolerance

5000 cells were seeded in 96-well plates for overnight growth, and then a series of concentrations of TMZ were added to wells containing LIF/CCL2 or PBS solvent. After 72h growth, cell viability was determined by the CCK-8 assay and IC50 was calculated using GraphPad Prism 5 software.

As can be seen in FIG. 3h, the IC50 value of LN18 cells for TMZ increased from 308.3(uM) to 500.9(uM), and the IC50 value of LN229 cells for TMZ increased from 237.8(uM) to 322.4(uM), after treatment with LIF/CCL 2. An increase in IC50 values indicates that LIF/CCL2 treatment promotes glioma cell tolerance to TMZ treatment. FIG. 3f prognostic data analysis shows LIF^H/CCL2^HThe patients who received the radiotherapy and chemotherapy had poor treatment effect and lower survival rate, which indicates that LIF^H/CCL2^HResulting in the tolerance of radiotherapy and chemotherapy and the reduction of the survival rate of patients. And LIF^L/CCL2^LThe curative effect of the patient is better, and the survival rate is higher. Simultaneous LIF^H/CCL2^HIs highly coincident with the survival curve of Mesenchymal subtype (mesnchymal) patients, and is consistent with our proposed conclusion that LIF/CCL2 can induce Mesenchymal subtype-associated phenotypes.

Example 3 ELISA testing of GBM patient serum LIF and CCL2, verifying that LIF/CCL2 expression correlates with mGBM.

The kit is purchased from R&D systems(

Human LIF Immunoassay # DLF00B and

human CCL2/MCP-1 Immunoassay # DCP00), sera of 7 patients (mGBM1, mGBM2 and 5 uGBM patients) were pretreated according to the manufacturer's instructions and then tested by ELISA. A standard curve was also established with recombinant human LIF and recombinant human CCL 2.

As can be seen in fig. 1f, the expression of LIF and CCL2 was significantly higher in the serum of 2 mGBM patients than in 5 uGBM patients. It is shown that the high expression of LIF and CCL2 can be used as a marker characteristic of mGBM patients, and the prognosis of mGBM patients is generally poor, so that the prognosis of patients can be predicted by detecting the serum expression of LIF and CCL 2.

Example 4 immunohistochemistry experiments confirmed poor prognosis in patients with high expression of LIF and CCL 2. And the expression correlation of LIF and CCL 2.

Paraffin section: and (3) placing the paraffin specimen into a refrigerator at the temperature of-20 ℃ for precooling, and then carrying out slicing, spreading, air drying and other steps.

Dewaxing: and (3) placing the paraffin sections in a 60 ℃ oven to be baked for 30min, and then carrying out a dewaxing step: xylene I15min, xylene II 15min, 100% alcohol 10min, 95% alcohol 5min, 85% alcohol 5min, 75% alcohol 5min, tap water 5min, PBS 5 min.

Antigen retrieval: the sections were rinsed thoroughly with PBS for 15 min. Preparing a repairing solution corresponding to the antibody, adding a proper amount of water into an autoclave, heating to boil, and placing the slices into the autoclave to be pressurized for 2min and 30 s. And immediately washing the cooling pressure cooker with tap water, taking out the slices, putting the slices into a repair box filled with repair liquid, and naturally cooling to room temperature.

Inactivation of endogenous catalase: taking out the slices, rinsing with PBS for 3 times, 5min each time, adding 3% H2O2, and reacting at 37 deg.C for 30 min.

And (3) sealing: the sections were removed and rinsed 3 times with PBS for 5min each time. After the section is gently spun off, the section is placed in a wet box, and goat serum sealer is dripped onto the tissue. Incubate in oven at 37 ℃ for 30 min.

Primary antibody incubation: spin-dry the sections gently, drip the diluted antibody onto the sections, cover the wet box lid, incubate overnight in a refrigerator at 4 ℃.

And (3) secondary antibody incubation: taking out the slices, rewarming to room temperature, placing the slices in a repair box, and rinsing with PBS for 3 times, 5min each time. After the PBS is gently spun off, the mixture is placed in a wet box, the immunohistochemical secondary antibody is dripped on the section, and the section is incubated in an oven at 37 ℃ for 30 min.

Color development: the wet box was removed and the sections were placed in a repair box and rinsed 3 times with PBS for 5min each time. Taking out the slices, placing the slices under a microscope, and dropwise adding DAB color development liquid. When the color developed to an appropriate degree, the reaction was immediately stopped by putting it in water.

Counterdyeing: washing the slices in tap water for 5min, staining in hematoxylin staining solution for 20s, washing with tap water, differentiating in hydrochloric acid alcohol for 1s, and washing with tap water until the slices turn blue.

Dehydrating and transparent: soaking the slices in 75% alcohol for 5min, 85% alcohol for 5min, 95% alcohol for 5min, 100% alcohol for 10min, xylene for 15min, and fresh xylene for 15 min.

Sealing: placing the slices in a fume hood for natural drying, dripping 50-100 μ L of neutral gum into each slice, and sealing with a cover glass. The slices can be placed in a 37 ℃ oven to remove air bubbles.

Scanning and storing: the slice scanner scans the slices and saves the images.

IHC staining evaluation was performed as follows according to our Immunohistochemical (IHC) evaluation system. The 5 most characteristic high power fields (400-fold magnification) for each tissue section were manually selected using an Olympus BX51 microscope (Olympus Tokyo Japan). The percentage of the signal positive tumor cells in all tumor cells is counted, and the positive rate classification standard is more than 50% (++), between 25% and 50% (+), between 5% and 25% (+), and less than 5% (-). Then, cases of "+++" and "++" were considered to be high expression of the target protein. Otherwise, cases are defined as low expression of the target protein.

As can be seen in FIG. 1d, we can distinguish patients into high-expression and low-expression groups of LIF and CCL2 by the intensity of positive signals of immunohistochemical staining. Further by analyzing the prognosis of the two groups of patients with high and low expression, as shown in FIG. 1e, it can be seen that the patients with high expression of either LIF, CCL2 or both LIF and CCL2 had worse prognosis than the patients with low expression. Therefore, the expression level of LIF and CCL2 can be detected by immunohistochemical staining, and the prognosis of the patient can be judged.

From FIG. 1a, we examined the expression of LIF and CCL2 in three different foci of an mGBM patient, and it can be seen that the expression of LIF and CCL2 are positively correlated, i.e. the foci CCL2 with high LIF expression is also high. Database analysis also showed the same results, as shown in FIG. 3c, the expression of LIF and CCL2 showed positive correlation.

Example 5 statistical analysis

GBM and glioma databases were downloaded from https:// xenoxybromiser. net/datapages/, and online analysis was done on gliovis. biolnfo. cnio. es/or http:// gepia2.cancer-pku. cn/# index website. DAVID bioinformatics resources and Gene Set Enrichment Analysis (GSEA) (P <0.05, FDR <0.25) was used to analyze our data. Statistical significance was determined by Student's t test, with P <0.05 considered statistically significant. The survival of the patients was evaluated using Kaplan-Meier survival plots and log-rank statistics. Relationships between different genes or proteins were analyzed by Pearson correlation data.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

1. A combination of gene targets for assessing the efficacy of a glioblastoma treatment comprising the CCL2 gene and the LIF gene.

2. Use of the combination of genetic targets of claim 1 in the preparation of a diagnostic reagent for assessing the efficacy of a glioblastoma treatment.

3. The use of claim 2, wherein the expression of CCL2 gene and LIF gene is detected.

4. The use of claim 2, wherein the expression of the CCL2 gene and LIF gene is determined by assaying mRNA or protein corresponding to the expression of the CCL2 gene and LIF gene.

5. The use of claim 2, for determining the clinical prognosis or treatment tolerance of glioblastoma.

6. A diagnostic reagent for evaluating the therapeutic effect of glioblastoma, comprising an agent for determining the expression of CCL2 gene and LIF gene.

7. The diagnostic reagent of claim 6, wherein the agent is selected from one or more of a primer, a probe, or an antibody.